Compositions and methods of targeting mutant K-Ras

ABSTRACT

The invention relates to a method of modeling K-Ras proteins with one or more mutations that result in constitutive activity, and identifying compounds that inhibit interactions among activated K-Ras proteins and their upstream and downstream effectors.

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 61/529,568 filed on Aug. 31, 2011. This and allother extrinsic materials discussed herein are incorporated by referencein their entirety. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

FIELD OF THE INVENTION

The described invention relates to computational investigation of thestructure of mutant K-Ras to permit rational design and discovery ofcompounds that specifically cause the destruction of cancer cellsbearing constitutively-activating mutations in K-Ras. More specifically,the invention relates to the discovery of a novel structural featureformed in K-Ras mutants and is targeted to inhibit the activity of themutant proteins.

BACKGROUND OF THE INVENTION

K-Ras (or Ki-Ras or Kirsten-Ras) is a 21 kD member of the Ras family ofGTPase proteins. Genetic alterations in the genome encoding for K-Rasare associated with development of neoplasia. Approximately 33% of allhuman tumors express mutant Ras, these mutations often stabilize Ras inGTP-bound (active) state. Mutations found in K-Ras associate stronglywith pancreatic cancer (90%), biliary tract cancer (33%), colorectalcancer (32%), and lung cancer (20%), among others. Approximately 20-25%of all human tumors harbor an activating mutation in gene encodingK-Ras. Examples of cancer-associated mutations are found at glycine-12(Gly12), Gly13, and glutamine-61 (Gln61), with Gly12 being thepredominant site of mutagenesis (88%).

Cancer-associated mutant K-Ras is constitutively active, with prolongedstabilization of its GTP-bound (active) state, and is thus able toconstitutively activate downstream client effectors such as Raf kinaseand phosphoinositide-3 kinase (PI3K). Both of these kinases playimportant roles in proliferation/survival/anti-apoptotic signalingpathways. These mutations have been implicated in insensitivity toEGFR-targeted anti-cancer therapies as mutations in K-Ras predisposecancer cells to be significantly less responsive to EGFR targetingtherapies (e.g., Panitumumab, Cetuximab, etc.). Interaction with the RasGTPase activating protein (RasGAP) is vital to the timely inactivationof K-Ras, resulting in more efficient hydrolysis of GTP to GDP. Theconformational changes in K-Ras structure stemming from GTP hydrolysisresult in the elimination of K-Ras' affinity for effector proteins,thereby inactivating downstream proliferation and anti-death pathways.Cancer-associated mutations in K-Ras have been shown to interact poorlywith RasGAP, therefore remaining in the “on” or constitutively activeposition.

In view of the important role K-Ras plays in various neoplastic diseasestates, it would be advantageous to be able to identify compounds thatbind specifically to the mutant K-Ras protein forms associated withcancer diseases states.

SUMMARY OF THE INVENTION

The present invention is drawn to compounds, compositions, and methodsof formation of complexes of a mutant K-Ras protein with a ligand. Inespecially preferred aspects, ligands and complexes are therapeuticallyeffective in the treatment of disorders that are associated with mutantK-Ras protein.

In one aspect of the inventive subject matter, the complex comprises aK-Ras protein (SEQ ID NO:1) with a G12V substitution, wherein theprotein with the G12V substitution has a first and a secondconformation, wherein the first conformation has a linear I-groovespanning residues Glu91, Asp92, Tyr96, Ala11, Gly60, Ala59, Thr35,Pro34, and Ile36, and wherein the second conformation has a branchedY-groove spanning Asp92, Ala11, Gln61, Glu62, Gly60, Ala59, Thr35,Arg68, Thr58, Tyr71 and Ile36, and wherein the compound bindsspecifically with the linear I-groove or Y-groove to form the complex.It is particularly preferred that the linear I-groove further comprisesa plurality of flanking residues selected from the group consisting ofGln61, Lys88, Val12, Glu62, and Tyr32, and that the branched Y-groovecomprises a selection of residues that includes Gln61, Ala11, Glu62,Ile36, Tyr71, Val12, Pro34 and Thr35. It is also contemplated that thecomplex further comprises one molecule of guanosine triphosphate (GTP)or analog thereof, typically with the groove encompassing theγ-phosphate of the GTP molecule. It is still further preferred that thecomplex is incapable of activating c-Raf or phosphoinositide 3-kinase(PI 3-kinase).

In particularly preferred complexes, the compound is selected from thegroup consisting of:1-[4-(4-{[2-(1H-imidazol-4-yl)ethyl]amino}piperidin-1-yl)phenyl]pyrrolidin-2-one,2-{[4-(4-acetylphenyl)piperazin-1-yl]methyl}-3H-quinazolin-4-one,1-methyl-3-[4-(4-{[2-(2-methylimidazol-1-yl)ethyl]amino}piperidin-1-yl)phenyl]imidazolidin-2-one,1-(2H-1,3-benzodioxol-5-ylmethyl)-4-[(3S)-1-(1H-imidazol-4-ylmethyl)piperidin-3-yl]piperazine,6-(aminomethyl)-2-(3-{[4-(pyrazol-1-ylmethyl)piperidin-1-yl]methyl}phenyl)-3H-pyrimidin-4-one,(4R)—N-[(6-chloroquinolin-2-yl)methyl]-1-(pyridin-2-yl)-4,5,6,7-tetrahydroindazol-4-amine,(3R,4R)-4-[4-(pyridin-2-yl)piperazin-1-yl]-1-(pyridin-2-ylmethyl)piperidin-3-ol,7-{[4-(4-acetylphenyl)piperazin-1-yl]methyl}pyrimido[2,1-b][1,3]thiazol-5-one,3-[(E)-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-phenylpyrazol-4-yl]methylidene}amino]-8-methyl-1,3-diazaspiro[4.5]decane-2,4-dione,1-(4-amino-1,2,5-oxadiazol-3-yl)-5-[4-(hydroxynitroso)phenyl]-N′-[(1E,2E)-3-phenylprop-2-en-1-ylidene]-1,2,3-triazole-4-carbohydrazide,3-({12-[4-(furan-2-ylcarbonyl)piperazin-1-yl]-8-oxo-15-oxa-14-azatetracyclo[7.6.1.0^{2,7}.0^{13,16}]hexadeca-1(16),2(7),3,5,9,11,13-heptaen-10-yl}amino)benzoicacid,4-chloro-N-[(7S)-7-(4-fluorophenyl)-5-phenyl-4H,7H-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl]benzenesulfonamide,(5S)-4-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)-5-(4-ethylphenyl)-3-hydroxy-1-(6-methyl-1,3-benzothiazol-2-yl)-5H-pyrrol-2-one,(5S)-1-(6-chloro-1,3-benzothiazol-2-yl)-4-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)-3-hydroxy-5-[4-(hydroxynitroso)phenyl]-5H-pyrrol-2-one,O-{[4-({4-[(2E)-2-[(3-fluorophenyl)methylidene]hydrazin-1-yl]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}amino)phenyl]nitroso}oxidanol,(2E)-3-(1,5-dimethyl-3-oxo-2-phenylpyrazol-4-yl)-5-[(3Z)-2-oxo-1H-indol-3-ylidene]-2-{2-[(3E)-2-oxo-1H-indol-3-ylidene]hydrazin-1-ylidene}-1,3-thiazolidin-4-one,5-(5-{[(4Z)-5-oxo-1,3-diphenylpyrazol-4-ylidene]methyl}furan-2-yl)-2H-isoindole-1,3-dione,5-[(6S)-5-benzyl-1H,4H,6H,7H-imidazo[4,5-c]pyridin-6-yl]-3-(pyridin-2-yl)-1,2,4-oxadiazole,2-(hydroxynitroso)-6-[(1E)-(2-{4-[(4-methylphenyl)amino]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}hydrazin-1-ylidene)methyl]phenol,(7′S)-12′-({[(5E)-1-methyl-2,4,6-trioxo-1,3-diazinan-5-ylidene]methyl}amino)-5′-phenyl-2′,5′-diazaspiro[1,5-diazinane-3,8′-tricyclo[8.4.0.0^{2,7}]tetradecane]-1′(14′),10′,12′-triene-2,4,6-trione,4-[(10R,10aR)-7,7-dimethyl-9,11-dioxo-6H,8H,10H,10aH-indeno[1,2-b]quinolin-10-yl]phenyl4-acetamidobenzenesulfonate,(5S)-1-(6-chloro-1,3-benzothiazol-2-yl)-4-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)-3-hydroxy-5-[3-(hydroxynitroso)phenyl]-5H-pyrrol-2-one,(5S)-4-(2,3-dihydro-1,4-benzodioxin-6-ylcarbonyl)-3-hydroxy-5-[3-(hydroxynitroso)phenyl]-1-(6-methyl-1,3-benzothiazol-2-yl)-5H-pyrrol-2-one,N-cyclooctyl-3-[(4-fluorobenzene)sulfonamido]-4-[(1S,9R)-6-oxo-7,11-diazatricyclo[7.3.1.0^{2,7}]trideca-2,4-dien-11-yl]benzamide,O-[(3-{[4,6-bis(1,2,3-benzotriazol-1-yl)-1,3,5-triazin-2-yl]amino}phenyl)nitroso]oxidanol,N-[4-({5,7-diphenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}sulfamoyl)phenyl]acetamide,3-({8-oxo-12-[4-(pyridin-2-yl)piperazin-1-yl]-15-oxa-14-azatetracyclo[7.6.1.0^{2,7}.0^{13,16}]hexadeca-1(16),2(7),3,5,9,11,13-heptaen-10-yl}amino)benzoicacid, and1-(4-amino-1,2,5-oxadiazol-3-yl)-N′-[(1Z)-3,4-dihydro-2H-naphthalen-1-ylidene]-5-phenyl-1,2,3-triazole-4-carbohydrazide.

In another aspect of the inventive subject matter, the complex comprisesa K-Ras protein (SEQ ID NO:1) with a G12D substitution, wherein theprotein has a conformation that comprises a groove spanning residuesAsp12, Ala59, Thr35, Asp57, Met67, Ile36, Ser39, Leu56, Thr58, Lys5,Lys16, Asp38, Val7 and Asp54, and a compound that binds specificallywith the groove to form the complex.

In a further aspect of the inventive subject matter, the complexcomprises a K-Ras protein (SEQ ID NO:1) with a G12C substitution,wherein the protein has a conformation that comprises a groove spanningresidues Cys12, Gln61, Glu62, Ala59, Gly60, Met67, Thr35, Ile36, Pro34,Tyr32, and a compound that binds specifically with the groove to formthe complex.

In a still further aspect of the inventive subject matter, the inventorsalso contemplate a method of identifying Ras effector interactioninhibitor compounds. Preferred methods include a step of providing amutant K-Ras protein, a step of forming a complex between a candidatecompound and a groove of the mutant K-Ras protein; and a further step ofdetermining the complex for binding affinity to a Ras effector, wherebythe candidate compound that forms the complex with a reduced bindingaffinity to the Ras effector is a Ras effector interaction inhibitor. Inespecially preferred methods, the mutant K-Ras protein comprises a G12V,G12D, or G12C mutation, and/or the Ras effector is a downstream effector(e.g., b-Raf, c-Raf or a PI 3-kinase). Alternatively, the Ras effectoris an upstream effector (e.g., EGFR or VEGFR). Suitable mutant proteinsmay be provided as an in silico construct (preferably achievingconformational stability after approximately 5 nanoseconds), or may bederived from a cancer associated with constitutive K-Ras activity (e.g.,biliary tract, bladder, breast, cervix, endometrial, kidney, largeintestine, liver, lung, melanoma, myeloid leukemia, ovarian, pancreas,and thyroid cancer).

Viewed from a different perspective, the inventors also contemplate amethod of forming a complex with a mutant K-Ras protein. Such methodswill preferably include a step of identifying a structural feature as abinding site in the mutant K-Ras protein, wherein the structural featureis selected from the group consisting of (a) a groove spanning residuesGlu91, Asp92, Tyr96, Ala11, Gly60, Ala59, Thr35, Pro34, and Ile36, (b) agroove spanning Asp92, Ala11, Gln61, Glu62, Gly60, Ala59, Thr35, Arg68,Thr58, Tyr71 and Ile36 (c) a groove spanning residues Cys12, Gln61,Glu62, Ala59, Gly60, Met67, Thr35, Ile36, Pro34, Tyr32, and (d) a groovespanning residues Asp12, Ala59, Thr35, Asp57, Met67, Ile36, Ser39,Leu56, Thr58, Lys5, Lys16, Asp38, Val7 and Asp54. In yet another step, apotential ligand is calculated for the structural feature by minimizingfee energy of the potential ligand to achieve a threshold value, and ina further step, the potential ligand is synthesized/obtained andcombined with the mutant K-Ras protein to thereby form the complex. Mostpreferably, the mutant K-Ras protein is a K-Ras protein (SEQ ID NO:1)with a G12V substitution, a K-Ras protein (SEQ ID NO:1) with a G12Dsubstitution, or a K-Ras protein (SEQ ID NO:1) with a G12C substitution.

Therefore, the inventors also contemplate a pharmaceutical compositionthat includes a pharmaceutically acceptable carrier and a mutant K-Rasprotein ligand that binds to a groove selected from the group consistingof (a) a groove spanning residues Glu91, Asp92, Tyr96, Ala11, Gly60,Ala59, Thr35, Pro34, and Ile36, (b) a groove spanning Asp92, Ala11,Gln61, Glu62, Gly60, Ala59, Thr35, Arg68, Thr58, Tyr71 and Ile36 (c) agroove spanning residues Cys12, Gln61, Glu62, Ala59, Gly60, Met67,Thr35, Ile36, Pro34, Tyr32, and (d) a groove spanning residues Asp12,Ala59, Thr35, Asp57, Met67, Ile36, Ser39, Leu56, Thr58, Lys5, Lys16,Asp38, Val7 and Asp54; wherein the mutant K-Ras protein ligand ispresent at a concentration effective to form a mutant K-Ras proteinligand complex in vivo.

While not limiting to the inventive subject matter, it is furtherpreferred that the pharmaceutical composition will also include a secondpharmaceutically active compound (e.g., a cytostatic or cytotoxiccompound), and/or a Ras inhibitor, a Raf inhibitor, a PI3K inhibitor, ora compound that re-activates p53 activity.

Consequently, the inventors also contemplate a method of treating apatient diagnosed with a neoplastic disease that is characterized by amutant K-Ras protein, wherein the method includes a step ofadministering to the patient contemplated pharmaceutical compositions,typically at a dosage and under a protocol to induce cell death in acell containing the mutant K-Ras protein.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows overlays of ribbon structures of molecular models of wildtype K-Ras and K-Ras mutants. In each Figure, the amino acid glycinelocated at position 12 (highlighted in dark gray) in wild type K-Ras hasbeen mutated to valine (A-B), aspartate (C), or cysteine (D) in theoverlay; glutamine at position 61 (white) is highlighted to illustratethe impact of mutations at position 12 on K-Ras' tertiary structure.Overlays of the wild type and G12V “I-groove” and G12V “Y-groove”structures are shown in (A) and (B), respectively. As illustrated in theFigure, the structures of the mutant and wild type proteins remainsrelatively well-aligned, with the exception of the residues in theso-called switch II region of K-Ras (residues 60-77), in which mutationsat the distant Gly12 alter the geometrical positioning of the switch IIamino acids, including Gln61. The positioning of Gln61 is thought to beimportant for interaction between KRas and RasGAP, the protein thatfacilitates inactivation of K-Ras.

FIG. 2 shows surface rendering of molecular models of the “Y-groove” and“I-groove” structures formed by the G12V mutant form of K-Ras, bothannotated to highlight amino acids that line the newly discoveredgrooves. The arrows indicate the locations of the ‘druggable’ groovesdiscovered to be specifically present in the G12V mutant. The RasGAPview represents the area of interaction between K-Ras and RasGAP, theprotein responsible for the inactivation of K-Ras via hydrolysis of GTPto GDP. The Effector view represents the area of interaction betweenK-Ras and its effector proteins (e.g., PI-3K, Raf, etc.).

FIG. 3A shows surface rendering of molecular models of the wild typeform of KRas, with the yellow oval indicating areas consisting of aminoacid residues which prohibit groove formation in the wild type. Thearrow indicates the location of the ‘druggable’ groove discovered to bespecifically present in the mutant K-Ras and absent in the wild type.The nucleotide and amino acid sequences for wild type K-Ras are shown inFIG. 3B (dark gray: Gly12, light gray: Gly13, medium gray: Gln61).

FIG. 4 shows surface rendering of molecular models of A) K-Ras G12D andB) K-Ras G12C, both annotated to illustrate amino acids that line thenewly discovered groove and pocket, respectively. The arrows in (A) andthe oval shading in (B) indicate the locations of the ‘druggable’ areasdiscovered to be specifically present in the G12D and G12C mutants,respectively.

FIG. 5 shows a RasGAP view (A) and an Effector view (B) of a surfacerendering of a sample model of a G12V mutant K-Ras with a bound moleculeof GTP and a candidate compound (A0383).

FIG. 6 illustrates the reduction of phosphorylation of MEK1 by compoundA0383(2-(hydroxynitroso)-6-[(1E)-(2-{4-[(4-methylphenyl)amino]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}hydrazin-1-ylidene)methyl]phenol)in 293 cells overexpressing K-Ras G12V. In (A), 10 M treatment withA0383 (sample 7) significantly reduced phosphorylation of MEK1 in anultra-sensitive immunoassay. (B) illustrates the lack of generaltoxicity in 293 cells (not overexpressing mutant K-Ras) in dosages ofA0383 ranging from 50-100 M.

DETAILED DESCRIPTION OF THE INVENTION

The presently described invention relates to the discovery of astructural feature present in mutant K-Ras proteins that was previouslyunreported. This structural feature provides a target against whichcandidate compounds are designed and screened, using computational andother screening methods.

The methods disclosed here use computational modeling of mutant K-Rasproteins to identify structural differences existing among wild type andmutant K-Ras proteins. Examples of cancer-associated mutations are foundat glycine-12 (Gly12), Gly13, and glutamine-61 (Gln61). Any of thesemutations, whether taken alone or in combination with one another, canbe used as the basis of an in silico model of mutant K-Ras proteins.Once one or more structural differences present in the mutant K-Rasproteins are identified, computational methods are then utilized toidentify compounds that bind to the specific structural feature.Alternatively, screening of compound libraries can be used to identifycompounds of interest.

A candidate compound is one that prevents or inhibits the binding of anactivated K-Ras protein or a mutant K-Ras protein with one or moreupstream or downstream effector proteins. Examples of upstream effectorsinclude the epithelial growth factor receptor (EGFR), platelet-derivedgrowth factor receptor (PDGFR), and the vascular endothelial growthfactor receptor (VEGFR). Examples of downstream effectors include b-Rafand c-Raf (Raf-1) which are serine/threonine-specific kinases and play arole in the MAPK/ERK signal transduction pathway, or a phosphoinositide3-kinase (PI 3-kinase).

Modeling

To date, the reported results of X-ray analysis of crystallized wildtype and mutant K-Ras have not shown significant structural differencesexisting between these forms of the protein. Because the staticstructures of the crystallized proteins failed to indicate structuraldifferences associated with the different functions of these proteins,alternative methods were employed, the results of which are discussedhere.

A molecular dynamics (MD) approach was used to explore the structures ofthe wild type and mutant K-Ras proteins. This approach resulted indetermining the predominant conformations of the wild type and G12V,G12C, and G12D mutant K-Ras proteins. It should be noted that any othermutation in the K-Ras amino acid sequence that produces constitutiveactivity can also be used, whether alone or in combination with othermutations, with the methods disclosed herein. The results of thisanalysis identified structural differences between the two proteins thatexplain the observed differences in functional behavior.

The molecular dynamics program Amber 11 was used for this structuralanalysis. The program is used to generate or provide in silicostructures of the mutant and wild type proteins. Amber 11 models thedynamic behavior of the in silico protein structures by calculating thedirection and magnitude of atomic forces acting on individual atoms,thereby providing a view of the conformational changes over biologicallyrelevant periods of time. Typically, structural conclusions can be madebased on 20-50 nanoseconds of simulated time, depending on the size andcomplexity of the system under study. A series of simulations was runusing the backbone conformation of the wild type K-Ras as the startingpoint. The G12V mutant structure was obtained from that of the wild typeprotein by replacing the side chain of Gly12 using molecular graphicssoftware. The Amber FF99SB force field was used to simulate bothproteins in explicit water. Modeling a protein in an explicitly definedvolume of water molecules gives the best possible accuracy, since itavoids the approximations that are made by continuum solvent models thattreat water as a continuous medium.

Using the described software and force field, the wild type and theG12V, G12D, and G12C mutants of K-Ras were simulated for over 500nanoseconds in several separate molecular dynamics simulations. Sinceeach simulation potentially explores conformational space along adifferent path, it is important to run several of them to maximizeconformational space coverage. Subsequent cluster analysis was used togroup observed protein conformations according to mutual similarity.Finally, representative structures were analyzed from the most abundantclusters. These structures correspond to the most common conformationsof each protein.

The overall structures of the wild type and the G12V, G12D, and G12Cmutant K-Ras were almost identical. However, notable differences weredetected in the region of a flexible loop spanning residues 60 through77 and frequently referred to as Switch II. The structures of the mutantand wild type proteins are provided as overlays in FIG. 1 for directcomparison. This Switch II loop is highly sensitive to local structuralchanges, including presence of GTP or GDP. The inherent flexibility ofSwitch II allows it to modulate properties of the surface used by K-Rasto bind its downstream effector proteins. In addition, residue Gln61located within Switch II has been implicated in the process of K-Rasinactivation by Ras GTPase activating protein (RasGAP). The Gln61residue of the wild type protein appears to provide charge stabilizationfor the interaction with Lys789 of RasGAP that is required for a stableK-Ras/RasGAP complex. With the complex correctly formed, GTP becomesreplaced with GDP, which inactivates Ras. However, G12V mutation ofK-Ras introduces a bulky valine side chain, which prevents the Gln61from maintaining its proper conformation.

Extensive molecular dynamics simulations revealed the exact nature ofthe resulting conformational changes. The mutant protein exists in twopredominant forms, which together account for approximately 80% of thetotal simulated time. These two conformations differ from the wild typeby the presence of a deep groove in the surface adjacent to the mutationsite. One of the mutant conformations features a linear groove that runsfrom Asp92 to Thr35 at the edge of the surface interface with b-Raf,PI3K, etc. (FIG. 2). This structural feature is referred to as theI-groove to reflect its geometry. The other conformation of the mutantK-Ras is similar to the I-groove conformation, but differs from it by abranching element in the surface groove that is directed toward Glu37.Since this feature has a branched topology, it was termed the Y-groove.None of these changes were observed in the simulations of the wild typeprotein. In addition, the wild type K-Ras remained completely stable forover 50 nanoseconds of simulated time.

The openings of both the I- and Y-grooves formed in the G12V mutantK-Ras are located on the edge of the surface of the protein responsiblefor binding of its effectors. Notably, two of the residues required forPI3K binding (Asp33 and Ile36) are located in the immediate proximity tothe groove openings. This structural feature made it possible tosimultaneously and specifically bind mutant K-Ras G12V and inhibit theinteraction between K-Ras G12V and PI3K, effecting the demise of cancercells expressing K-Ras G12V.

FIG. 2 shows side and top down views, of the I-groove and Y-groovestructures formed by G12V mutant K-Ras with GTP bound (indicated inyellow). The I-groove on the surface of K-Ras G12V is defined by Glu91,Asp92, Tyr96, Ala11, Gly60, Ala59, Thr35, Pro34, and Ile36. It is alsobounded by Gln61, Lys88, Val12 and Glu62. The Y-groove is outlined byAsp92, Ala11, Gln61, Glu62, Gly60, Ala59, Thr35, Arg68, Thr58, Tyr71.The side chain of Ile36 serves as the divider between the two branchesof the Y-groove. In comparison, FIG. 3 shows a side view and a top viewof the wild type protein. Without wishing to be bound by theory, itappears that the side chains of Gly12, Gln61 and other residues in thearea of the groove serve to protect or prevent the groove from forming.Accordingly, mutation of other residues in this general area of theK-Ras protein may give rise to other constitutively active K-Rasvariations.

Molecular dynamics simulations that were used for modeling of the G12Vmutant of K-Ras can be used equally successfully to model other mutantsof this protein. We have carried out a series of simulations of othermutated variants of K-Ras predominant in a variety of human tumors,including G12C and G12D.

Each in silico protein structure was prepared by using the backbone ofthe wild type K-Ras and replacement of the side chain in question.Simulations were run for 50 ns and each produced several dominantconformations, which differ from the structure of the wild type K-Ras.

The G12D mutant of K-Ras developed a deep groove spanning its surfaceaway from the GTP binding site along the effector binding surface (FIG.4A). The residues that define this groove are Asp12, Ala59, Thr35,Asp57, Met67, Ile36, Ser39, Leu56, Thr58, Lys5, Lys16, Asp38, Val7 andAsp54. Due to its close proximity to the effector binding surface, thisgroove represents a suitable target for computational discovery ofactive compounds targeting specifically the G12D mutant of K-Ras.

The G12C mutant features a similar structural element, which differs inits overall geometry, but is located in approximately the same part ofthe protein structure (FIG. 4B). The wider and more defined pocket ofthe G12C mutant occupies the following residues: Cys12, Gln61, Glu62,Ala59, Gly60, Met67, Thr35, Ile36, Pro34, Tyr32. It also is flanked bythe GTP molecule. The overall shape and location of this pocket presentsan opportunity to specifically target the G12C mutant of K-Ras.

The model system described above is equally applicable to otherconstitutively active mutations of K-Ras, including, but not limited to,G12R, G12S, G12A, G13D, G13C, Q61H, Q61L, and Q61R.

Contemplated Compounds

The methods described herein have yielded a number of compounds thathave been modeled to interact with the groove in the mutant K-Rasprotein G12V. Molecular docking software was used to facilitatediscovery of compounds capable of binding the described I- and Y-groovesand blocking the interaction of Asp33 and Ile36 with PI3K. Moleculardocking was based on the two dominant conformations of K-Ras produced byour molecular dynamics simulations and led to the discovery of novelcompounds that specifically bind mutant K-Ras G12V, do not efficientlybind wild type K-Ras, and disrupt the interaction of K-Ras G12V withPI3K.

Molecular docking simulations started with ˜1 million compounds selectedfrom a larger set to satisfy the constraints of size, functionality andphysical properties. The initial docking has produced a set ofprotein-ligand complexes ranked by estimated affinity. This stage wasfollowed by a high accuracy scoring approach based on molecular dynamicsand referred to as MM-PBSA (Molecular Mechanics-Poisson-BoltzmannSurface Area). MM-PBSA offers the possibility of simulating a dynamicsequence of states for each complex in aqueous solution. Eachprotein-ligand complex was simulated by molecular dynamics in explicitwater, and statistical analysis of the structure was performed. MM-PBSAscores were then used to improve the accuracy of compound ranking andselect candidates for experimental testing.

An illustration of a typical modeling experiment is shown in FIGS. 5Aand 5B. These drawings show the mutant G12V K-Ras protein with Val12 indark red. A candidate compound is colored by element (carbon is gray,oxygen is red, and nitrogen is blue), and a molecule of GTP is shown inorange.

Some of the discovered compounds are presented:

Compound Name Structure Score 1-[4-(4-{[2-(1H-imidazol-4-yl)ethyl]amino}piperidin-1- yl)phenyl]pyrrolidin-2-one Reference #A0158

2-{[4-(4-acetylphenyl)piperazin-1- yl]methyl}-3H-quinazolin-4-one A0190

1-methyl-3-[4-(4-{[2-(2- methylimidazol-1- yl)ethyl]amino}piperidin-1-yl)phenyl]imidazolidin-idin-2-one Reference # A0159

1-(2H-1,3-benzodioxol-5- ylmethyl)-4-[(3S)-1-(1H-imidazol-4-ylmethyl)piperidin-3- yl]piper-azine Reference # A0161

6-(aminomethyl)-2-(3- {[4-(pyrazol-1-ylmethyl)piperidin-1-yl]methyl}phenyl)-3H-pyrimidin-4-one Reference # A0162

(4R)-N-[(6-chloroquinolin-2- yl)methyl]-1-(pyridin-2-yl)-4,5,6,7-tetrahydroindazol-4-amine Reference # A0163

(3R,4R)-4-[4-(pyridin-2- yl)piperazin-1-yl]-1-(pyridin-2-ylmethyl)piperidin-3-ol Reference # A0164

7-{[4-(4-acetylphenyl)piperazin-1-yl]methyl}pyrimido[2,1-b][1,3]thiazol-5-one Reference # A0188

3-[(E)-{[3-(2,3-dihydro-1,4-benzodioxin-6-yl)-1- −40.20phenylpyrazol-4-yl]methylidene}amino]-8-methyl-diazaspiro[4.5]decane-2,4-dione Reference # A04641-(4-amino-1,2,5-oxadiazol-3-yl)-5-[4- −33.55(hydroxynitroso)phenyl]-N′-[(1E,2E)-3-phenylpropen-1-ylidene]-1,2,3-triazole-4-carbohydrazide Reference # A04783-({12-[4-(furan-2-ylcarbonyl)piperazin-l-yl]-8- −31.55 oxo-15-oxa-14-azatetracyclo[7.6.1.0{circumflex over ( )}{2,7}.0{circumflex over( )}{13,16}]hexadeca- 1(16),2(7),3,5,9,11,13-heptaen-10-yl}amino)benzoic acid Reference # A05064-chloro-N-[(7S)-7-(4-fluorophenyl)-5-phenyl- −31.444H,7H-[1,2,4]triazolo[1,5-a]pyrimidin-2- yl]benzenesulfonamide Reference# A0452 (5S)-4-(2,3-dihydro-1,4-benzodioxin-6- −31.25ylcarbonyl)-5-(4-ethylphenyl)-3-hydroxy-1-(6-methyl-1,3-benzothiazol-2-yl)-5H-pyrrol-2-one Reference # A0453(5S)-1-(6-chloro-1,3-benzothiazol-2-yl)-4-(2,3- −30.74dihydro-1,4-benzodioxin-6-ylcarbonyl)-3-hydroxy-5-[4-(hydroxynitroso)phenyl]-5H-pyrrol- 2-one Reference # A0474O-{[4-({4-[(2E)-2-[(3- −30.26 fluorophenyl)methylidene]hydrazin-1-yl]-6-(morpholin-4-yl)-1,3,5-triazin-2- yl}amino)phenyl]nitroso}oxidanolReference # A0490 (2E)-3-(1,5-dimethyl-3-oxo-2-phenylpyrazol-4- −30.18yl)-5-[(3Z)-2-oxo-1H-indol-3-ylidene]-2-{2-[(3E)-2-oxo-1H-indol-3-ylidene]hydrazin-1-ylidene}-1,3-thiazolidin-4-one Reference # A04915-(5-{[(4Z)-5-oxo-1,3-diphenylpyrazol-4- −30.18ylidene]methyl}furan-2-yl)-2H-isoindole-1,3- dione Reference # A04975-[(6S)-5-benzyl-1H,4H,6H,7H-imidazo[4,5- −29.69c]pyridin-6-yl]-3-(pyridin-2-yl)-1,2,4-oxadiazole Reference # A02042-(hydroxynitroso)-6-[(1E)-(2-{4-[(4- −29.17methylphenyl)amino]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}hydrazin-1-ylidene)methyl]phenol Reference # A0383(7′S)-12′-({[(5E)-1-methyl-2,4,6-trioxo-1,3- −28.98diazinan-5-ylidene]methyl]amino)-5′-phenyl-2′,5′-diazaspiro[1,5-diazinane-3,8′- tricyclo[8.4.0.0{circumflex over( )}{2,7}]tetradecane]-1(14′),10′,12′- triene-2,4,6-trione Reference #A0455 4-[(10R,10aR)-7,7-dimethyl-9,11-dioxo- −28.716H,8H,10H,10aH-indeno[1,2-b]quinolin-10- yl]phenyl4-acetamidobenzenesulfonate Reference # A0500(5S)-1-(6-chloro-1,3-benzothiazol-2-yl)-4-(2,3- −28.43dihydro-1,4-benzodioxin-6-ylcarbonyl)-3-hydroxy-5-[3-(hydroxynitroso)phenyl]-5H-pyrrol- 2-one Reference # A0481(5S)-4-(2,3-dihydro-1,4-benzodioxin-6- −27.96ylcarbonyl)-3-hydroxy-5-[3- (hydroxynitroso)phenyl]-1-(6-methyl-1,3-benzothiazol-2-yl)-5H-pyrrol-2-one Reference # A0482N-cyclooctyl-3-[(4-fluorobenzene)sulfonamido]- −27.834-[(1S,9R)-6-oxo-7,11- diazatricyclo[7.3.1.0{circumflex over( )}{2,7}]trideca-2,4-dien-11- yl]benzamide Reference # A0507O-[(3-{[4,6-bis(1,2,3-benzotriazol-1-yl)-1,3,5- −27.60triazin-2-yl]amino}phenyl)nitroso]oxidanol Reference # A0484N-[4-({5,7-diphenyl-[1,2,41triazolo[1,5- −27.16a]pyrimidin-2-yl}sulfamoyl)phenyl]acetamide Reference # A03463-({8-oxo-12-[4-(pyridin-2-yl)piperazin-1-yl]-15- −26.67 oxa-14-azatetracyclo[7.6.1.0{circumflex over ( )}{2,7}.0{circumflex over( )}{13,16}]hexadeca- 1(16),2(7),3,5,9,11,13-heptaen-10-yl}amino)benzoic acid Reference # A03791-(4-amino-1,2,5-oxadiazol-3-yl)-N′-[(1Z)-3,4- −25.98dihydro-2H-naphthalen-1-ylidene]-5-phenyl-1,2,3-triazole-4-carbohydrazide Reference # A0508

Compounds specifically inhibiting the G12V mutant K-Ras proteinconformations fall into two broad categories—those targeting theI-groove and the compounds that bind to the Y-groove. The former have alinear structure with abundant hydrophobic groups, such as phenyl rings,which provide the necessary rigidity and render the compound relativelyflat. The latter are branched compounds designed to occupy the entireavailable volume of the Y-groove. They also consist mostly of aromaticrings and have multiple heteroatom-containing functional groups, whichtend to improve aqueous solubility. Typically, there are few, if any,hydrogen bonds within the protein-ligand complexes, and most of thebinding free energy is derived from hydrophobic interactions that aremaximized by shape complementarity.

In order to confirm specificity of the discovered compounds toward themutant forms of K-Ras, we have performed molecular docking of theselected ligands to the wild type K-Ras and scored the obtained boundorientations with MM-PBSA, following the same standard protocol. Nearlyall scores were worse than those calculated for the ligand complexeswith the mutant protein. The range of MM-PBSA scores was between −23.27and −8.3, with the majority of compounds being at worse than −20. Thisfurther justifies the score cutoff for compound selection set at −25.Those few compounds that exhibited scores between −20 and −25 are boundto the wild type K-Ras in locations that are distant from the effectorbinding surface and are highly unlikely to influence normal function ofthe protein even if the binding in vivo is significant.

Additional compounds are identified using standard techniques well knownto those of ordinary skill in the art. For example, high throughputscreening of compounds that block or inhibit binding of mutant K-Rasproteins with known downstream proteins such as b-Raf, c-Raf (Raf-1) orPI 3-kinase. Alternatively, compound libraries can be generated usingstandard techniques well known to those of ordinary skill in the artusing the compounds disclosed herein as starting material.

Exemplary Uses of Contemplated Compounds

The compounds identified by the methods described herein are useful forthe treatment of cancers associated with mutant K-Ras activity.Approximately 33% of all human tumors express mutant Ras, thesemutations often stabilize Ras in the GTP-bound (active) state. Mutationsfound in K-Ras associate strongly with pancreatic cancer (90%), biliarytract cancer (33%), colorectal cancer (32%), and lung cancer (20%),among others. Approximately 20-25% of all human tumors harbor anactivating mutation in gene encoding K-Ras. Examples ofcancer-associated mutations are found at glycine-12 (Gly12), Gly13, andglutamine-61 (Gln61), with Gly12 being the predominant site ofmutagenesis (88%). Exemplary cancers associated with constitutive K-Rasactivity include but are not limited to biliary tract, bladder, breast,cervix, endometrial, kidney, large intestine, liver, lung, melanoma,myeloid leukemia, ovarian, pancreas, and thyroid cancer.

The invention provides a pharmaceutical composition comprising describedcompounds and at least one pharmaceutically acceptable excipient orcarrier. Methods of preparing such pharmaceutical compositions typicallycomprise the step of bringing into association a described compound witha carrier and, optionally, one or more accessory ingredients. Thedescribed compounds and/or pharmaceutical compositions comprising samemay be formulated into pharmaceutically-acceptable dosage forms byconventional methods known to those of skill in the art. Typically,formulations are prepared by uniformly and intimately bringing intoassociation a described compound with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions of the present invention suitable forparenteral administration comprise one or more described compounds incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Pharmaceutical formulations of the described compounds suitable for oraladministration may be in the form of capsules, cachets, pills, tablets,lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofa described compound as an active ingredient. A described compound mayalso be administered as a bolus, electuary or paste. A describedcompound may be administered transdermally (e.g., using a patch).

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular described compound employed, orthe ester, salt or amide thereof, the route of administration, the timeof administration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the described compounds employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

In general, a suitable daily dose of a described compound will be thatamount of the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the describedcompounds for a patient, when used for the indicated effects, will rangefrom about 1 mcg to about 5 mg per kilogram of body weight per hour. Inother embodiments, the dose will range from about 5 mcg to about 2.5 mgper kilogram of body weight per hour. In further embodiments, the dosewill range from about 5 mcg to about 1 mg per kilogram of body weightper hour.

If desired, the effective daily dose of a described compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In one embodiment, the describedcompound is administered as one dose per day. In further embodiments,the compound is administered continuously, as through intravenous orother routes. In other embodiments, the compound is administered lessfrequently than daily, such as every 2-3 days, in conjunction withdialysis treatment, weekly or less frequently.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration. As used herein, theterm “route” of administration is intended to include, but is notlimited to subcutaneous injection, subcutaneous depot, intravenousinjection, intravenous or subcutaneous infusion, intraocular injection,intradermal injection, intramuscular injection, intraperitonealinjection, intratracheal administration, intraadiposal administration,intraarticular administration, intrathecal administration, epiduraladministration, inhalation, intranasal administration, oraladministration, sublingual administration, buccal administration, rectaladministration, vaginal administration, intracisternal administrationand topical administration, transdermal administration, oradministration via local delivery (for example by catheter or stent).The described compounds may also be administered or co-administered inslow release dosage forms. The disclosed compounds have efficacy whenadministered systemically.

The following examples are offered to illustrate but not to limit theinvention.

Example 1: Structural Analysis of Wild Type and Mutant K-Ras

The molecular dynamics program Amber 11 was used to analyze wild typeand mutant K-Ras for structural differences. A series of simulations wasrun using the backbone conformation of the wild type K-Ras as thestarting point. The G12V, G12D, and G12C mutant structures were obtainedfrom that of the wild type protein by replacing the side chain of Gly12using molecular graphics software. The Amber FF99SB force field was usedto simulate mutant proteins in explicit water.

An initial premise for this study was that, given the differentfunctional behavior exhibited by the mutant and wild type K-Ras proteinsin vivo, there should be one or more structural difference evidentbetween these proteins. Because no such differences were detected usingcrystallographic methodologies, molecular dynamic methods were selectedto discover the structural reasons for such a difference. Allsimulations were started from the identical protein structures with theonly exception of the mutation at the 12 position in the peptide chain.In this way, there was no pre-existing bias toward any particularprotein conformation.

The simulations were run for over 100 nanoseconds of simulated time ineach run, with noticeable differences between the G12V mutant, G12Dmutant, G12C mutant, and the wild type K-Ras emerging after about first10 nanoseconds. The remainder of the simulation served to confirm thatthe differences were not random fluctuations, but rather stable featuresdue to the different side chains at the position 12.

Analysis of the resulting structures of the wild type and conformationalforms of the mutant K-Ras has revealed major differences in the shape ofthe protein surface near the GTP γ-phosphate. The G12V mutant exists insolution as an ensemble of conformations, with two of them being themost predominant and accounting for at least 80% of the total simulatedtime. One of the forms is characterized by a deep groove in the surfacethat stretched for about 16 Å from Asp92 to Ile36. This groove in themutant K-Ras appears to have originated from the dramatic change in theposition of Gln61 side chain, which swings away from the bulky isopropylgroup of the Val12 in the mutant K-Ras. The other conformer of themutant K-Ras features a branched variant of the surface groove termedthe Y-groove, which refers to the geometry of this surface feature. Bothopenings of the Y-groove are on the interaction surface of K-Ras withPI3K and Raf. The wild type protein has no such features, and apparentlyhas a shallow groove that runs in perpendicular direction from Lys117 toTyr64. In the wild type protein, the Gln61 points toward the glycine atthe 12 position and is generally located in proximity of the GTPγ-phosphate.

The I-groove on the surface of K-Ras spans the following set ofresidues: Glu91, Asp92, Tyr96, Ala11, Gly60, Ala59, Thr35, Pro34, andIle36. It is also flanked by side chains of the following: Gln61, Lys88,Val12, Glu62, Tyr32 and the γ-phosphate of the GTP. The Y-groove isdefined by the following residues: Asp92, Ala11, Gln61, Glu62, Gly60,Ala59, Thr35, Arg68, Thr58, Tyr71. The side chain of Ile36 serves as thedivider between the two branches of the Y-groove.

The overall shape and size of both grooves makes them capable ofaccommodating an organic drug-like compound. In addition, the groovesopen near Ile36 and Pro34, the contact residues important for formationof the complex with RAF and p110 subunit of PI3K. If this interaction isinhibited by a compound that targets the groove, downstream signaling bymutant K-Ras will be disrupted. At the same time, the wild type proteinwill be unaffected.

Example 2: In Vitro Cell Death Assays

The validity of the current computational approach is confirmed bytesting the ability of the candidate compounds to inhibit the functionof constitutively-active mutant K-Ras. Inhibition of mutant K-Rasexpression has previously been shown to specifically activate the demiseof mutant expressing cancer cells (Zhu et al., Canc. Biol. & Ther.,2006; Chen et al., World J. Gastro, 2005; Zhang et al., Canc. Res.,2006). Thus the following methodology is used to confirm the predictedefficacy of the identified compounds.

Protocol for Cell Death Assessment:

Screening compounds that are predicted to selectively target K-Rasmutant cells will comprise of assessment of cell death in human cancercells bearing activating K-Ras G12V mutations (e.g., human colon SW480)and counter assessment of viability in K-Ras WT human cells (e.g., HT29,293, etc.). SW480 and HT29 cells are both propagated at 37° C./5% CO₂and in RPMI+10% fetal bovine serum (FBS) and DMEM+10% FBS, respectively.Upon plating cells in microtiter plates, compounds are supplemented atvarious concentrations and viability is assessed within 24-72 hrs bymanual or automated (Countess (Invitrogen)) trypan blue cytometry.

Cell death analysis of candidate compound A0383 in K-Ras wild typeexpressing 293 cells did not uncover unspecific toxicity (IC₅₀>100 μM)(FIG. 6B).

Protocol for Assessment of Alterations in Protein Biochemistry:

Compounds predicted to interfere with the function of mutant K-Ras willimpact downstream signaling mechanisms, including though not limited tothe K-Ras/PI3K/Akt/mTOR and K-Ras/Raf/MEK/ERK pathways. Screening forinhibition of constitutive activation of these pathways will consist ofdetermining the phosphorylation patterns indicative of activation ofdownstream effector proteins. Specifically, the phosphorylation eventsapparent in Akt and MEK 1 or 2 kinases are assessed for the impact ofcompounds in the K-Ras/PI3K/Akt/mTOR and K-Ras/Raf/MEK/ERK pathways,respectively. Briefly, wild type and mutant K-Ras expressing cells aretreated with compounds for 24-48 hrs, cells collected and lysed, andproteins separated via standard electrophoresis techniques.Alternatively, 293 cells overexpressing WT, G12V, G12D, G12C, or G13 aretreated with compounds for 24-48 hours, cells collected and lysed, andproteins separated via standard electrophoresis. Alternatively, lysedcells are subjected to ultra-sensitive immunoassays. Finally,immunoreactivity with phospho-specific antibodies to Akt and MEK1/2determines impact on the pathways mentioned above.

Immunoassay with candidate compound A0383 showed significant reductionin the phosphorylation and activation of the downstream Ras signalingprotein MEK, which is phosphorylated by the Ras effector protein Raf(FIG. 6A).

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

The invention claimed is:
 1. A mixture comprising: a K-Ras protein (SEQID NO:1) with a G12V substitution, wherein the K-Ras protein with theG12V substitution has a first and a second mutant conformation, whereinthe first mutant conformation has a first mutant groove defined byGlu91, Asp92, Tyr96, Ala11, Gly60, Ala59, Thr35, Pro34, and Ile36;wherein the second mutant conformation has a second mutant grooveoutlined by Asp92, Ala11, Gln61, Glu62, Gly60, Ala59, Thr35, Arg68,Thr58, and Tyr71; and a compound bound to the first or second groove,wherein the compound is2-(hydroxynitroso)-6-[(1E)-(2-{4-[(4-methylphenyl)amino]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}hydrazine-1-ylidene)methyl]phenol) (A0383).